1. Field of the Invention
The present invention relates to a method and device for the simultaneous concentration of multiple waterborne pathogens from large volumes of liquid toxins, and more particularly to the effective concentration of small numbers of multiple waterborne pathogens from large volumes of various water matrices.
2. Description of the Related Art
Effective concentration of small numbers of multiple waterborn pathogens from large volumes of various water matrices is a serious challenge. Current commercially available technologies are not able to automatically concentrate and elute simultaneously multiple pathogens from large liquid volumes using portable equipment. Pathogens size, variability and biological differences requires the use of specific, expensive, and labor intensive concentration techniques, especially for viruses and protozoa.
Currently, the two major approaches for concentrating multiple microorganisms from large water volumes include filtration and continuous flow centrifugation. Ultrafilters, which organisms are retained by size exclusion have much smaller pore size than viruses, which allow for the simultaneous concentration of viruses, bacteria, and protozoa. The particles are kept in the retentate and thus prevent clogging of the filter surface. Several publications have described the application of hollow fiber ultrafilters for concentrating human viruses, see Bicknell, 1985, Cryptosporidium oocysts (Simmons, 2001; Kuhn and Oshima, 2002, Ferguson, 2004), and the simultaneous concentration of viruses, bacteria and protozoa (Juliano and Sobsey, 1997/1998; Morales-Morales, 2003; Hill 2005).
However, ultrafiltration methodology is cumbersome and requires further systematic evaluation for much larger volumes of water than the 100 L volumes that are currently available. Electropositive filters are not rated by pore size and were initially designed to capture viruses from large water volumes by electrostatic attraction. There have been a few publications that evaluated their efficacy to concentrate bacteria and protozoa. See Hou et al, (1980) which reported substantially high retention of E. coli, Viruses (Polio, and MS2 bactenophages, and endotoxin) by using charged modified filters. Watt et al. (2002) reported low recoveries for Cryptosporidium oocysts, Giardia cysts and Polio virus from large water volumes, 14% and 17%, respectively.
Recently, two configurations of the ZetaPlus® Virosorb® 1 MDS filter were evaluated for simultaneous recovery of multiple microorganism types from tap water (Polaczky et al., 2007). High numbers of C. parvum, S. enterica and B. globigii, and bacteriophages, were spiked into 25 L of tap water samples. Low recoveries were achieved using the cartridge filter, 21%, 37%and 87%, respectively and much higher recoveries, 28%, 50%, 65%, respectively, were reported for the flat filter.
Continuous flow centrifugation (CFC) enables large scale collection of particles, such as protozoa and bacteria, through their sedimentation due to high centrifugal forces. Whitmore and Carrington, (1993) employed a bench-top centrifuge for the recovery of C. parvum oocysts from water samples.
Utilization of stationary bench-top blood cell separators with recoveries of several folds higher than the cartridge filtration was also reported (Goatcher, 1996). Substantially high recoveries (>90%) were reported for simultaneous concentration of Cryptosporidium, Giardia, Microsporidium, and bacteria from small volumes of source and potable water using a stationary centrifuge and a costly labor-intensive protocol (Borchardt and Spencer 1996; Borchardt and Spencer, 1998; Borchardt and Spence 2002).
However, less efficient recoveries of C. parvum oocysts from 100 L were also reported by this method (Swales and Wright, 2000). In addition, very low recoveries, less than 5%) of C. parvum from 10 L of water spiked source water, using a compact continuous flow centrifuge were reported (Higgins, 2003).
The above reports demonstrate that currently suggested approaches for concentrating multiple microbe types from large water samples are limited by manually operated systems, using expensive disposables and employing tedious protocols that are not suitable for rapid response to accidental or deliberate water contamination and may complicate routine monitoring.
An earlier prototype of a modified blood separation apparatus, such as the 625B standard disposable bowl manufactured by Haemonetics, of Braintree, Mass.) efficiently concentrated Cryptosporidium oocysts, Giardia cysts, and Microsporidian spores from tap and source water samples. The basic principle underlying the Continuous Flow Centrifugation (CFC) technique has also been described (Zuckerman et al, 1999; Zuckerman and Tzipori, 2004; 2006).
However, there still exists the need for a continuous flow centrifuge that enables concentration and elution of targets from large volumes of liquids.